Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. TSE infectivity can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding TSE pathogenesis and developing effective therapeutics. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrP-res or PrPSc) of the normal, protease-sensitive prion protein, PrP-sen. Susceptibility to infection can be influenced by amino acid homology between PrP-sen and PrP-res while differences in structure between PrP-res molecules from different prion strains are believed to encode strain phenotypes. My studies address many different aspects of prion diseases at both the molecular and pathogenic level. In particular, my laboratory focuses on: 1) identifying the earliest events which occur during prion infection, 2) precisely defining the different cellular compartments where PrP-res formation occurs, 3) determining the molecular basis of prion strains and, 4) development of effective prion therapeutics. Prion diseases are diseases of protein folding. Determining the structure of PrP-res is therefore critical for understanding not only how a normal host protein can become infectious but also for determining whether or not different PrP-res structures are responsible for encoding different prion strains. Most protein structure techniques require highly purified protein. However, PrP-res preparations are contaminated with multiple other proteins (Moore, R.A., Timmes, A., Wilmarth, P.A., and Priola, S.A. (2010). Comparative profiling of highly enriched 22l and Chandler mouse scrapie prion protein preparations. Proteomics 10: 2858-2869) which might confound the analysis of PrP-res structure. In 2011, we utilized a LC-MS/MS Nanospray Ion Trap Mass Spectrometer and an improved PrP-res purification technique to study whether or not other molecules that co-purify with PrP-res can contribute to the structural analysis of PrP-res from different prion strains. Our results show that non-PrP proteins do contribute to alpha helical, loop/turn, and beta sheet structures that previously had been solely attributed to PrP-res. Furthermore, since our improved PrP-res purification can in some cases remove greater than 99% of the major non-PrP contaminants, our data suggest that the alpha helical, loop/turn, and beta sheet secondary structure that remains are derived from PrP-res itself. Our study is the first to utilize a combined proteomics/protein structure approach for analyzing PrP-res conformation. In 2011, we completed in vivo work for the initial studies analyzing acute prion infection in mice and are currently in the process of analyzing the data. We also initiated in vitro studies characterizing the interaction of PrP-res with the cell during the initial stages of both mouse and human prion infection.